Project Summary/Abstract: Alcoholic liver disease (ALD) is one of the main causes of chronic liver disease worldwide and accounts for up to 48% of deaths associated with end stage liver disease in the United States 1. Compared to other types of liver disease, there has been little progress in the pharmacological management of ALD. Factors contributing to a lack of specific therapeutic targets include current suboptimal disease models and the gap in our knowledge of mechanisms of susceptibility and tolerance to chronic alcohol exposure. We propose to bridge this gap by using human stem cell and gene editing technology to study the most important genetic susceptibility factor involved in ALD progression; the risk variant polymorphism of PNPLA3 2-4. The variant allele of PNPLA3 (148 isoleucine to methionine protein variant; I148M) induces abnormal lipolysis and contributes to enhanced hepatic steatosis as a result of chronic alcohol consumption, as well as more aggressive progression to inflammation and fibrosis that are characteristic of the advanced stages of ALD2,4. We have already generated the human induced pluripotent stem cell lines that will be used for this proposal, and we have optimized a protocol to differentiate these stem cells to hepatocytes. We have performed basic characterization of PNPLA3 variant hepatocytes and confirmed that they accumulate triglyceride-rich lipid droplets and express inflammatory cytokines that are thought to be involved in progression from steatosis to hepatitis. To understand the mechanism by which the variant PNPLA3 confers increased susceptibility to alcohol-related liver injury, we will perform detailed profiling of lipid droplets of hepatocytes derived from human stem cell lines engineered to express the risk variant of PNPLA3. We will determine the lipid composition, protein composition and profile of specific inflammatory mediators that determine progression from steatosis to inflammation in ALD patients. We will use ethanol and palmitic acid to mimic the environmental stress induced by alcohol consumption and lipotoxicity. Our main goal is to identify novel therapeutic targets directly in live human hepatocytes that can be exploited for the treatment of ALD.